介绍一种简便的红细胞无氧糖酵解率测定方法

目的为了简化现有的华伯氏呼吸仪检测红细胞无氧糖酵解的方法,进行此项研究.方法实验用葡萄糖试剂盒,高氯酸,可见光分光光度计,氮气瓶和摇床.实验分4步进行任氏液.Trls-HCI溶液等试液的配制,任氏血的制备,红细胞无氧糖酵解率的测定和红细胞无氧糖酵解率的计算.结果用SOD保养液.在4℃条件下保存入红细胞75d时糖酵解率为86.2%±5.0%,明显优于GMA保养液39.2%±8.9%.结论不用华伯氏仪的红细胞无氧糖酵解率的测定方法,操作简便,方法准确可靠,在普通实验室即可进行.     

SOD对4℃保存人红细胞损伤的防护作用研究

目的：探讨SOD对延时保存红细胞损伤防护作用的机理。方法：取义务献血人员静脉血,置4℃冰箱保存。保存前后不同时期用标准方法分别检查各项指标,用生物荧光素标记红细胞,测定24h活存率。实验分3组：ACD和／或GMA、抗氧化剂（SOD）组。结果：SOD组保存75d,无氧糖酵解率维持在保存前的86．2％,ATP为56．4％,PK为64．3％,明显高于GMA或ACD组。保存75d整体回输后24h活存率为86．1％,与GMA保存42d(89.1%)结果接近。结论：用抗氧化剂保养液在4℃条件下保存人血红细胞从42d延长到75d,红细胞无氧糖酵解率、ATP、PK和整体回输后24h活存率等均与42d保存方相符,为红细胞的活存提供了理论根据。     

生物酶法制浆半纤维素酶液的防腐技术研究

研究结果表明,在室温（30±5℃）下,乙酸乙酯、苯甲酸钠对放线菌z-6所产半纤维素酶液有防腐作用,酶液保存10d,酶活剩余50％左右;5．0％的氯化钠对青霉菌m8所产半纤维素酶液有明显防癌作用,酶液保存15d,酶活剩余57．7％。     

双歧杆菌对窒息新生儿红细胞免疫功能的影响

目的 :研究双歧杆菌活菌制剂对窒息新生儿红细胞免疫功能的影响。方法 :窒息新生儿 38例随机分为金双歧治疗组 (简称治疗组 ) 2 0例和非金双歧治疗组 (简称对照组 ) 1 8例。于生后 2 4h内采血检测红细胞C3b受体花环率 (E C3bRR)和红细胞免疫复合物花环率 (E ICR) ,随后予以脱水、止惊等常规治疗 ,治疗组加金双歧 (双歧杆菌活菌制剂 )口服 7d ,并于第 7～ 8天各组再次抽血进行上述检测。结果 :生后第 1天两组新生儿E C3bRR和E ICR差异无显著性 (P >0 0 5) ;7～ 8d时治疗组E C3bRR为 (1 9 2 0±4 1 2 ) %显著高于对照组 (1 2 2 7%± 3 63 % ,p <0 0 5) ,E ICR为 (1 1 53± 3 1 8) %显著低于对照组(1 5 40 %± 3 0 2 % ,p <0 0 5)。 结论 :双歧杆菌可使红细胞免疫功能上调 ,从而增强窒息新生儿的抗感染能力和减轻脑的缺血性损伤     

缺血预处理合并低温及晶体停搏液对兔未成熟心肌的保护作用

本文拟探讨缺血预处理(ischemic preconditioning,IP)合并低温及晶体停搏液对幼兔的离体心脏是否具有心肌保护作用.采用Langendorff离体心脏灌注模型,灌注液为Krebs-Henseleit液(K-H液).取3～4周龄幼兔心脏,在第一部分实验中分为Con、IP1、IP2、IP3组(n=6),分别给予0、1、2、3次IP,其后各组心脏均在20℃低温下停灌2 h,37℃常温下再灌注30 min.在第二部分实验中分为SConI、SCon2、SCon3、SIPl、SIP2、SIP3组(n=8),其中SIPl、SIP2、SIP3组给予2次IP后灌注St.Tho-mas Ⅱ晶体停搏液(CCS)使心脏停搏,然后分别使心脏在32℃、25℃、20℃下停灌30、90和120min,其后各组均在37℃再灌注30 min.SConl,SCon2,SCon3三组则不给予IP,继续灌注20min后灌注CCS使心脏停搏,然后分别在32℃、25℃、20℃下停灌30、90和120 min,其后各组均在37℃再灌注30 min.以Maclab/4 s生理实验系统记录平衡末、缺血前、再灌注后1、3、5、10、20、30 min时心率(HR)、左心室发展压(LVDP)以及左心室内压上升及下降最大速率(±dp/dtmax),测定再灌注末心肌组织中ATP和丙二醛(MDA)的含量,以及超氧化物歧化酶(SOD)的活性.在20℃低温停灌且停灌期间不给予CCS时,再灌注末IP2组LVDP×HR、+dp/dtmax和-dp/dtmax的恢复率分别为96%±21%、101%±19%和84%±15%,显著高于Con组和IP3组(P＜0.01,P＜0.05);心肌组织的ATP含量亦高于Con组(P＜0.01).在不同低温停灌且停灌期间给予CCS时,再灌注末SIP1、SIP2组的LVDP×HR、+dp/dtmax分别恢复到87%±14%、99%±26%(P＜0.05,vs SConl group)和87%±16%、102%±20%(P＜0.05,vs SCon2 group);心肌组织的ATP含量均分别显著高于SCon1组和SCon2组(P＜0.05),心肌组织MDA含量亦分别低于SCon1组和SCon2组(P＜0.05).上述结果提示,IP对在20℃低温停灌的兔未成熟心脏具有一定的心肌保护作用,2次IP的保护效应优于1次或3次IP.在停灌期间应用CCS,IP的心肌保护作用随停灌期间低温程度的升高而减弱.     

不同降温速率对脐血干细胞冷冻复苏后生物学特性的影响

考察了不同降温速率对脐血造血干细胞各种生物学特性的影响。在4℃～-40℃的降温范围内,分别选择-0.5℃/min, -1℃/min, -5℃/min的降温速率进行降温,对复苏后的脐血单个核细胞的回收率、活性和CD34+含量的变化以及BFU-E、CFUGM和CFU-MK集落的回收率进行了考察,发现在-1℃/min的降温速率下,脐血MNC回收率可达93.3%±1.8%,活性可达95.0%±3.9%, CD34^＋细胞回收率达80.0%±17.9%,BFUE回收率为87.1%±5.5%,CFUGM回收率达88.5%±8.9%,CFUMK的回收率也达到86.2%±7.4%。并且对复苏后的细胞进一步进行体外培养,发现在-1℃/min的降温速率下复苏的细胞仍然具有与未经冷冻细胞相似的扩增能力,而-0.5℃/min和-5℃/min这两种降温速率条件下复苏的细胞与未经冷冻的细胞相比差距较大。因而-1℃/min的降温速率对冻存脐血干细胞比较合适。     

金针菇多糖的提取工艺优化及荧光标记研究

通过响应面法优化金针菇多糖热水浸提的最佳工艺,并用异硫氰酸荧光素(FITC)标记金针菇多糖(FVP),研究标记多糖的体外稳定性和细胞毒性。结果表明:优化的最佳浸提条件为液料比41∶1(m L∶g)、提取温度92℃、提取时间3 h,金针菇多糖的提取率为4.87%;通过还原胺化反应实现了金针菇多糖的FITC标记,FITC的取代度为0.90%,在24 h内标记的金针菇多糖体外稳定性良好,且不影响FVP的抑瘤活性。     

HCV编码病毒结构蛋白的DNA免疫研究

应用HCVC (pC)和HCVCE1E2 (pCE1E2 )重组体转染真核细胞并且通过肌注免疫BALB/C小鼠 ,对其体液免疫和细胞免疫进行检测。所用的 pC和 pCE1E2 均可在真核细胞内表达出特异性HCVC蛋白 ;肌注DNA免疫后均可诱导出BALB/C小鼠的体液和细胞免疫反应 ,抗体反应的A值 :pC组为 0 .358± 0 .0 96 ,pCE1E2 组为 0 .4 15± 0 .12 7;CTL活力 pC组为 18.6 5%± 5.72 % ,pCE1E2 组为 2 0 .0 7%± 11.11% ;通过免疫鼠荷瘤检测体内CTL反应 ,可观察到免疫组鼠发瘤时间滞后 ,发瘤部位减少和存活时间延长。在开发和研制HCV疫苗的过程中 ,DNA免疫是在快速构建、评价和筛选免疫原方面的有效办法。     

双斑恩蚜小蜂的生殖方式及其在烟粉虱体内的发育

首次对双斑恩蚜小蜂Encarsia bimaculata Heraty et Polaszek雌蜂的胚胎发育全过程进行连续观测和数码摄像,明确了双斑恩蚜小蜂的生殖方式是两性产雌,雌蜂为初寄生蜂,孤雌产雄且雄蜂为自复寄生。雌蜂在温度(25±1)℃,相对湿度70%±10%条件下,从卵到成虫的发育历期为(13.1±2.5)d,卵期(1.98±0.38)d,幼虫1龄(0.95±0.33)d,2龄(2.48±0.40)d,3龄(3.46±0.43)d,预蛹(0.95±0.20)d,蛹(4.88±0.80)d。     

布氏田鼠冷暴露中褐色脂肪组织的增补及解偶联蛋白基因表达

布氏田鼠 (Microtusbrandti)随机分组暴露在冷环境 [12L∶12D ,(4± 2 )℃ ]中 12h ,1d ,3d ,7d ,14d ,2 1d和 2 8d ;对照组生活在温暖环境下 [12L∶12D ,(2 5± 2 )℃ ]。与对照组相比 ,布氏田鼠的褐色脂肪组织 (BAT)重量在冷暴露 12h～ 3d时降低 ,7～ 2 1d时则增加。 7～ 2 8d冷暴露组动物的BAT总蛋白和总DNA含量均比对照组明显提高。冷环境中的布氏田鼠解偶联蛋白 (UCP)的mRNA随时间的延长而表达上调 ,在冷暴露 2 1d时达到高峰。结果表明 ,冷暴露能够诱导布氏田鼠BAT细胞增补和UCP基因表达 ,从而使适应性产热增加。     

微流控芯片技术在细胞生物学研究中的应用进展

20世纪90年代以来,微流控芯片技术得到了快速发展。由于具有小型化、集成化、高通量、低消耗、分析快速等特点,微流控芯片作为一种新型的生物学研究平台,能够提供传统方法不具备的精细和可控制的细胞研究条件,在细胞生物学研究领域中得到了广泛关注。该文主要介绍其在细胞培养、分选、裂解、计数、凋亡检测、迁移、单细胞捕获、细胞间作用等方面的研究进展。     

Efficient and Innocuous Live‐Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry

The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry. Herein, the author reviews pioneering examples of such new cell‐based assays, provides evidence that challenges the paradigm that cell penetration is a necessarily damaging and stressful event for cells, and highlights some of the challenges that should be addressed to fully unlock the potential of this nascent field.     

Ultrastructural evidence for two-cell and three-cell neural pathways in the tentacle epidermis of the sea anemone Aiptasia pallida.

Sensory and ganglion cells in the tentacle epidermis of the sea anemone Aiptasia pallida were traced in serial transmission electron micrographs to their synaptic contacts on other cells. Sensory cell synapses were found on spirocytes, muscle cells, and ganglion cells. Ganglion cells, in turn, synapsed on sensory cells, spirocytes, muscle cells, and other neurons and formed en passant axo-axonal synapses. Axonal synapses on nematocytes and gland cells were not traced to their cells of origin, i.e., identified sensory or ganglion cells. Direct synaptic contacts of sensory cells with spirocytes and sensory cells with muscle cells suggest a local two-cell pathway for spirocyst discharge and muscle cell contraction, whereas interjection of a ganglion cell between the sensory and effector cells creates a local three-cell pathway. The network of ganglion cells and their processes allows for a through-conduction system that is interconnected by chemical synapses. Although the sea anemone nervous system is more complex than that of Hydra, it has similar two-cell and three-cell effector pathways that may function in local responses to tentacle contact with food.     

The role of glutamine, serum and energy factors in growth of enterocyte-like cell lines

Background: Glutamine is routinely added to most cell cultures. Glutamine has been found to be the preferential nutrient to the rapidly replicating intestinal mucosa, but whether this is a metabolic effect or due to other properties of this amino acid is not determined. To study the importance of glutamine on the growth of two enterocyte-like cell lines, the effects of depriving the media or supplementing it with glutamine were assessed in media with different serum and energy supplements. Methods: CaCo-2 and HT-29 cells were grown in serum-free medium, with fetal bovine or synthetic serum, and with or without glucose or galactose. The glutamine content was varied between 0 and 4 mM. All growth assays were performed in triplicate by counting in a hemocytometer. Results: Both cell lines were dependent of serum factors for growth, but displayed distinct requirements on glutamine supplementation. Glutamine was an obligate supplement with dose-dependent correlation to growth (r=0.87, p<0.01) for CaCo-2 cells cultured in synthetic, but not in fetal bovine serum. In HT-29 cells, the correlation between glutamine and growth was significant (r=0,68, p<0,05) only in fetal bovine serum in the absence of galactose. Conclusion: This study shows that glutamine has different growth stimulating effects on two enterocyte-like cell lines studied. This could reflect different modes of action of glutamine on proliferation and differentiation in an enterocyte cell population.     

Of mice, frogs and flies: generation of membrane asymmetries in early development

Embryonic development begins with cleavage of the fertilized egg. Cleavage comprises two major processes: cytokinesis and formation of a polarized epithelial cell layer. The focus of this review is comparison of the generation of membrane polarity during embryonic cleavage in three different developmental model systems. In mammalian embryos, as exemplified by analysis of the mouse, generation of distinct membrane domains is uncoupled from cleavage divisions and is initiated in a specific developmental phase, called compaction. In Xenopus laevis embryos, generation of polarized blastomeres occurs simultaneously with cytokinesis. The origin of specific membrane domains of X. laevis polar blastomeres, however, can be traced back to oogenesis. Finally, in Drosophila melanogaster, generation of polarized cells occurs at cellularization. The relevance of cell adhesion, cell junctions and cytocortical scaffolds will be discussed for each of the model systems. Despite enormous morphologic differences, the three models share many common features; in particular, many important molecular interactions are conserved.     

哺乳动物体细胞核移植中供体细胞的研究进展

在哺乳动物体细胞核移植中,供体细胞是影响其效率的主要因素之一。供体细胞的类型、细胞周期、细胞的培养代数、冷藏与冷冻处理,以及供体动物的性别、年龄等都可能影响核移植胚胎的发育。根据现有资料,简要综述了在哺乳动物体细胞核移植中有关供体细胞的研究进展。     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

Engineering Cardiac Cell Junctions In Vitro to Study the Intercalated Disc

Abstract

This review article discusses a recent work using engineered cardiac cells to study the function of the intercalated disc putting emphasis on mechanical and electrical coupling.     

Mammalian testis: a target of in vivo electroporation

Mammalian spermatogenesis consists of three biologically significant processes: stem cell self-renewal and differentiation, meiosis, and haploid cell morphogenesis. Understanding the molecular mechanisms behind these processes might provide clues to the puzzle of species preservation and evolution, and to treatments for male infertility. However, few useful in vitro systems exist to investigate these processes at present. To elucidate these mechanisms, in vivo electroporation of the testis might be a convenient option. Since DNA solution can be injected into the seminiferous tubule via the rete testis, similar to germ cell transplantation, it is easy to transfect expression vectors into various differentiated germ cells and supporting Sertoli cells with adequate electric shock. Unfortunately, it is difficult to create transgenic animals using this method because of its low efficiency. However, gain- and loss-of-function assays, promoter assays, and tagged-protein behavior assays can be conducted with this technique, as in in vitro culture systems.     

Perivascular cells for regenerative medicine

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.